CDunham-September 2008

Using the Spectrophotometer to Introduce the Process of Photosynthesis

Background Information

Light is composed of particles called photons that act like waves. Visible light energy is a

very small fraction of the electromagnetic spectrum pictured below. Changes in the

wavelength of visible light result in a change of color. The colors of visible light

commonly referred to as ROYGBIV correspond to differing wavelengths.

Source: http://www.dnr.sc.gov/ael/personals/pjpb/lecture/spectrum.gif

The color of an object is largely due to the way those objects interact with light and

ultimately reflect or transmit it to our eyes. A blue molecule appears blue because it is

reflecting the blue wavelengths of light. Colored molecules present in a solution will

selectively absorb, reflect or transmit certain wavelengths of light. A spectrophotometer

can be used to measure the amount of light that is transmitted or absorbed by a molecule

within a solution.

Objectives

Colored molecules within a solution interact with light waves to either absorb or reflect

certain wavelengths of light.

The color of an object is an indication of the wavelengths of light that are being reflected

from the object

Observations of the Visible Light Spectrum

Obtain a spectroscope and colored acetate squares from your teacher.

http://www.chem.uiuc.edu/Chem103/spectroscopy/images/simplified_spectroscope.png

CDunham-September 2008

1. Look through the spectroscope towards the light. List the colors of light you see

within the spectroscope and their respective wavelengths in your laboratory

notebook.

2. Remove the colored acetate squares from your envelope. Position one of the

colored acetate squares directly over the slit of the spectroscope. Compare the

spectrum obtained coming through each colored acetate with the spectrum you

observed from step one. Note any similarities or differences in your laboratory

notebook.

Using a Spectrophotometer to Obtain the Absorbance Spectra of Colored Solutions

Source: http://www.rlc.dcccd.edu/mathSci/reynolds/MICRO/2420/spectrophotometer.jpg

1. Turn on the spectrophotometer by turning dial number two to the right.

2. Let the machine warm up for 20 minutes.

3. Set the wavelength to 400 nm by turning dial number one.

4. Set the machine to transmittance by pushing the mode button (3).

5. Set the spectrophotometer to zero percent transmittance by turning dial number

two.

6. Insert the tube containing distilled water into the cuvette holder (4) and close the

lid.

*Wipe all fingerprints from test tube

*Insert tube so that the line on the tube is lined up with the front line of the

cuvette holder (4)

7. Set the spectrophotometer to one hundred percent transmittance by turning dial

number five.

8. Remove distilled water tube from cuvette holder (4)

9. Insert one of the test tubes containing colored water into the cuvette holder (4).

*Wipe all fingerprints from test tube

*Insert tube so that the line on the tube is lined up with the front line of the

cuvette holder (4)

10. Set the machine to absorbance by pushing the mode button (3).

11. Record the absorbance in your laboratory notebook.

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CDunham-September 2008

12. Increase the wavelength (dial number 1) by 10 nm and record the absorbance.

13. Repeat in 10nm increments, recording the absorbance each time, until wavelength

reaches 660 nm

14. Repeat with alternate colors

15. Graph data

Data Analysis

Record the answers to the questions below in your laboratory notebook

1. What wavelengths of light were absorbed for the

a) Green solution

What colors of visible light do these wavelengths correspond to?

b) Red Solution

What colors of visible light do these wavelengths correspond to?

c) Blue Solution

What colors of visible light do these wavelengths correspond to?

2. What wavelengths of light were transmitted for the

a) Green solution

What colors of visible light do these wavelengths correspond to?

b) Red Solution

What colors of visible light do these wavelengths correspond to?

c) Blue Solution

What colors of visible light do these wavelengths correspond to?

3. What wavelengths of light do you think plants use to make their food?

4. Look at the absorbance spectrum below. What color do you think the solution

was? Why?

Source: http://www.che.wsu.edu/home/modules/96modules/Gabler/images/transparency1.gif